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Retinoic acid (RA) induces spermatogonial differentiation, but the mechanism by which it operates remains largely unknown. We developed a germ cell culture assay system to study genes involved in spermatogonial differentiation triggered by RA. Stimulated by RA 8 (Stra8), a RA-inducible gene, is indispensable for meiosis initiation, and its deletion results in a complete block of spermatogenesis at the pre-leptotene/zygotene stage. To interrogate the role of Stra8 in RA mediated differentiation of spermatogonia, we derived germ cell cultures from the neonatal testis of both wild type and Stra8 knock-out mice. We provide the first evidence that Stra8 plays a crucial role in modulating the responsiveness of undifferentiated spermatogonia to RA and facilitates transition to a differentiated state. Stra8-mediated differentiation is achieved through the downregulation of a large portfolio of genes and pathways, most notably including genes involved in the spermatogonial stem cell self-renewal process. We also report here for the first time the role of transcription elongation regulator-1 like (Tcerg1l) as a downstream effector of RA-induced spermatogonial differentiation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Approximately 80% of childhood cancers can now be cured but a side effect of treatment results in about one-third of the surviving boys being infertile or severely subfertile when they reach reproductive age. Currently, more than 1 in 5000 men of reproductive age who are childhood cancer survivors suffer from this serious quality of life problem. It is possible to obtain a testicular biopsy before treatment to preserve the spermatogonial stem cells (SSCs) of the male by cryopreservation, but the results of long-term storage of SSCs on their subsequent functional ability to generate normal offspring has not been examined in any mammalian species. Moreover, it will be necessary to increase the number of these cryopreserved SSCs to remove any contaminating malignant cells and assure regeneration of spermatogenesis., Methods and Results: In this report, we demonstrate that long-term cryopreservation (>14 years) of testis cells from mouse, rat, rabbit and baboon safeguards SSC viability, and that these cells can colonize the seminiferous tubules of recipient testes. Moreover, mouse and rat SSCs can be cultured and re-establish complete spermatogenesis, and fertile mouse progeny without apparent genetic or epigenetic errors were generated by the sperm produced., Conclusions: These findings provide a platform for fertility preservation in prepubertal boys undergoing gonadotoxic treatments.

Insight regarding mechanisms controlling gene expression in the spermatogonial stem cell (SSC) will improve our understanding of the processes regulating spermatogenesis and aid in treating problems associated with male infertility. In the present study, we explored the global gene expression profiles of the glial cell line-derived neurotrophic factor (GDNF)-regulated transcription factors Ets (E-twenty-six) variant gene 5 (Etv5); B-cell chronic lymphocytic leukemia (CLL)/lymphoma 6, member B (Bcl6b); and POU domain, class-3 transcription factor 1 (Pou3f1). We reasoned that these three factors may function as a core set of transcription factors, regulating genes responsible for maintaining the SSC population. Using transient siRNA oligonucleotides to individually target Etv5, Bcl6b, and Pou3f1 within mouse SSC cultures, we examined changes to the global gene expression profiles associated with these transcription factors. Only modest overlaps in the target genes regulated by the three factors were noted, but ETV5 was found to be a critical downstream regulator of GDNF signaling that mediated the expression of several known SSC self-renewal related genes, including Bcl6b and LIM homeobox 1 (Lhx1). Notably, ETV5 was identified as a regulator of Brachyury (T) and CXC chemokine receptor, type 4 (Cxcr4), and we showed that ETV5 binding to the Brachyury (T) gene promoter region is associated with an active state of transcription. Moreover, in vivo transplantation of SSCs following silencing of Brachyury (T) significantly reduced the number of donor cell-derived colonies formed within recipient mouse testes. These results suggest Brachyury is of biological importance and functions as part of GDNF/ETV5 signaling to promote self-renewal of mouse SSCs cultured in vitro.

MicroRNAs (miRs) play a key role in the control of gene expression in a wide array of tissue systems, where their functions include the regulation of self-renewal, cellular differentiation, proliferation, and apoptosis. However, the functional importance of individual miRs in controlling spermatogonial stem cell (SSC) homeostasis has not been investigated. Using high-throughput sequencing, we profiled the expression of miRs in the Thy1(+) testis cell population, which is highly enriched for SSCs, and the Thy1(-) cell population, composed primarily of testis somatic cells. In addition, we profiled the global expression of miRs in cultured germ cells, also enriched for SSCs. Our results demonstrate that miR-21, along with miR-34c, -182, -183, and -146a, are preferentially expressed in the Thy1(+) SSC-enriched population, compared with Thy1(-) somatic cells. Importantly, we demonstrate that transient inhibition of miR-21 in SSC-enriched germ cell cultures increased the number of germ cells undergoing apoptosis and significantly reduced the number of donor-derived colonies of spermatogenesis formed from transplanted treated cells in recipient mouse testes, indicating that miR-21 is important in maintaining the SSC population. Moreover, we show that in SSC-enriched germ cell cultures, miR-21 is regulated by the transcription factor ETV5, known to be critical for SSC self-renewal.

The development of techniques to maintain the spermatogonial stem cell (SSC) in vivo and in vitro for extended periods essentially allows for the indefinite continuation of an individual germline. Recent evidence indicates that the aging of male reproductive function is due to failure of the SSC niche. SSCs are routinely cultured for 6 mo, and no apparent effect of culture over this period has been observed. To determine the effects of SSC aging, we utilized an in vitro culture system, followed by quantitative transplantation experiments. After culture for 6 mo, SSCs that had been aged in vivo for 1500 days had a slower proliferation rate than SSCs that were aged in vivo to 8 or 300 days. Examination of methylation patterns revealed no apparent difference in DNA methylation between SSCs that were aged 8, 300, or 1500 days before culture. Long-term culture periods resulted in a loss of stem cell potential without an obvious change in the visual appearance of the culture. DNA microarray analysis of in vivo- and in vitro-aged SSCs identified the differential expression of several genes important for SSC function, including B-cell CLL/lymphoma 6, member B (Bcl6b), Lim homeobox protein 1 (Lhx1), and thymus cell antigen 1, theta (Thy1). Collectively, these data indicate that, although both in vitro and in vivo aging are detrimental to SSC function, in vitro aging results in greater loss of function, potentially due to a decrease in core SSC self-renewal gene expression and an increase in germ cell differentiation gene expression.

In the human testis, beginning at approximately 2 months of age, gonocytes are replaced by adult dark (Ad) and pale (Ap) spermatogonia that make up the spermatogonial stem cell (SSC) pool. In mice, the SSC pool arises from gonocytes approximately 6 days after birth. During puberty in both species, complete spermatogenesis is established by cells that differentiate from SSCs. Essentially pure populations of prepubertal human spermatogonia and mouse gonocytes were selected from testis biopsies and validated by confirming the presence of specific marker proteins in cells. Stem cell potential of germ cells was demonstrated by transplantation to mouse testes, following which the cells migrated to the basement membrane of the seminiferous tubule and were maintained similar to SSCs. Differential gene expression profiles generated between germ cells and testis somatic cells demonstrated that expression of genes previously identified as SSC and spermatogonial-specific markers (e.g., zinc-finger and BTB-domain containing 16, ZBTB16) was greatly elevated in both human spermatogonia and mouse gonocytes compared to somatic cells. Several genes were expressed at significantly higher levels in germ cells of both species. Most importantly, genes known to be essential for mouse SSC self-renewal (e.g., Ret proto-oncogene, Ret; GDNF-family receptor alpha1, Gfr alpha1; and B-cell CLL/lymphoma 6, member B, Bcl6b) were more highly expressed in both prepubertal human spermatogonia and mouse gonocytes than in somatic cells. The results indicate remarkable conservation of gene expression, notably for self-renewal genes, in these prepubertal germline cells between two species that diverged phylogenetically approximately 75 million years ago.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis throughout the life of a male. Because SSCs of many species can colonize the mouse testis, and glial cell line-derived neurotrophic factor (GDNF) is responsible for stimulating SSC self-renewal in rodents, we reasoned that molecular mechanisms of SSC self-renewal are similar across species. GDNF-regulated genes have been identified in mouse SSCs; however, downstream targets of GDNF are unknown in other species. The objective of this work was to identify GDNF-regulated genes in rat SSCs and to define the biological significance of these genes for rat SSC self-renewal. We conducted microarray analysis on cultured rat germ cells enriched for SSCs in the presence and absence of GDNF. Many GDNF-regulated genes were identified, most notably, Bcl6b and Etv5, which are important for mouse SSC self-renewal. Bcl6b was the most highly regulated gene in both the rat and mouse. Additionally, we identified three novel GDNF-regulated genes in rat SSCs: Bhlhe40, Hoxc4, and Tec. Small interfering RNA treatment for Bcl6b, Etv5, Bhlhe40, Hoxc4, and Tec resulted in a decrease in SSC number, as determined by transplantation, without a change in total cell number within the culture. These data indicate that, like in the mouse SSC, Bcl6b and Etv5 are important for rat SSC self-renewal, suggesting that these genes may be important for SSCs in all mammals. Furthermore, identification of three novel GDNF-regulated genes in the rat SSC extends our knowledge of SSC activity and broadens the foundation for understanding this process in higher species, including humans.

Self-renewal and differentiation of spermatogonial stem cells (SSCs) provide the foundation for testis homeostasis, yet mechanisms that control their functions in mammals are poorly defined. We used microarray transcript profiling to identify specific genes whose expressions are augmented in the SSC-enriched Thy1(+) germ cell fraction of mouse pup testes. Comparisons of gene expression in the Thy1(+) germ cell fraction with the Thy1-depleted testis cell population identified 202 genes that are expressed 10-fold or higher in Thy1(+) cells. This database provided a mining tool to investigate specific characteristics of SSCs and identify novel mechanisms that potentially influence their functions. These analyses revealed that colony stimulating factor 1 receptor (Csf1r) gene expression is enriched in Thy1(+) germ cells. Addition of recombinant colony stimulating factor 1 (Csf1), the specific ligand for Csf1r, to culture media significantly enhanced the self-renewal of SSCs in heterogeneous Thy1(+) spermatogonial cultures over a 63-day period without affecting total germ cell expansion. In vivo, expression of Csf1 in both pre-pubertal and adult testes was localized to clusters of Leydig cells and select peritubular myoid cells. Collectively, these results identify Csf1 as an extrinsic stimulator of SSC self-renewal and implicate Leydig and myoid cells as contributors of the testicular stem cell niche in mammals.